Cereal starch comprises two types of molecule, amylose and amylopectin. Amylose is an essentially linear molecule composed of α-1,4 linked glucosidic units, while amylopectin is highly branched with α-1,6 glucosidic bonds linking linear chains.
The synthesis of starch in the endosperm of higher plants is carried out by a suite of enzymes that catalyse four key steps. Firstly, ADP-glucose pyrophosphorylase (ADGP) activates the monomer precursor of starch through the synthesis of ADP-glucose from G-1-P and ATP. Secondly, the activated glucosyl donor, ADP-glucose, is transferred to the non-reducing end of pre-existing α-1,4 linked chains by starch synthases. Thirdly, starch branching enzymes (SBE) introduce branch points through the cleavage of a region of α-1,4 linked glucan followed by transfer of the cleaved chain to an acceptor chain, forming a new α-1,6 linkage. SBEs are the only enzymes that can introduce the α-1,6 linkages into α-polyglucans and therefore play an essential role in the formation of amylopectin. Finally, starch debranching enzymes remove some of the branch linkages, although the mechanism through which they act is unresolved (Myers et al., 2000). While it is clear that at least these four activities are required for normal starch granule synthesis in higher plants, multiple isoforms of each of the four activities are found in the endosperm of higher plants and specific roles have been proposed for individual isoforms on the basis of mutational analysis (Wang et al, 1998; Buleon et al., 1998) or through the modification of gene expression levels using transgenic approaches (Abel et al., 1996, Jobling et al., 1999, Schwall et al., 2000). However, the precise contributions of each isoform of each activity to starch biosynthesis are still not known, and it is not known whether these contributions differ markedly between species.
In the cereal endosperm, two isoforms of ADP-glucose pyrophosphorylase are present, one form within the amyloplast, and one form in the cytoplasm (Denyer et al., 1996, Thorbjornsen et al., 1996). Each form is composed of two subunit types. The shrunken (sh2) and brittle (bt2) mutants in maize represent lesions in large and small subunits respectively (Giroux and Hannah, 1994). Four classes of starch synthase are found in the cereal endosperm, an isoform exclusively localised within the starch granule, granule-bound starch synthase (GBSS), two forms that are partitioned between the granule and the soluble fraction (SSI, Li et al., 1999a, SSII, Li et al., 1999b) and a fourth form that is entirely located in the soluble fraction, SSIII (Cao et al, 2000, Li et al., 1999b, Li et al. 2000). GBSS has been shown to be essential for amylose synthesis (Shure et al., 1983), and mutations in SSII and SSIII have been shown to alter amylopectin structure (Gao et al, 1998, Craig et al., 1998). The rice GBSS (waxy) gene sequence has been described (Wang et al., 1990), and expression inhibited by antisense methods (Terada et al., 2000). The waxy gene is expressed in endosperm and pollen but not in other rice organs (Hirano and Sano, 2000).
Two main classes of SBEs are known in plants, SBEI and SBEII. SBEII can further be categorized into two types in cereals, SBEIIa and SBEIIb (Boyer and Preiss, 1978; Gao et al., 1996; Fisher et al., 1996; Hedman and Boyer, 1982; Mizuno et al., 1992; Sun et al., 1997; Sun et al., 1998). Additional forms of SBEs are also reported in some cereals, the putative 149 kDa SBEI from wheat (Baga et al., 2000) and the 50/51 kDa SBE from barley (Sun et al., 1996). Genomic and cDNA sequences have been characterized for rice (Nakamura and Yamanouchi, 1992; Mizuno et al, 1992; Mizuno et al, 1993; Mizuno et al, 2001), maize (Baba et al., 1991; Fisher et al., 1993; Gao et al., 1997) wheat (Repellin et al., 1997; Nair et al., 1997; Rahman et al., 1997) and other cereals. Sequence alignment reveals a high degree of sequence similarity at both the nucleotide and amino acid levels and allows the grouping into the SBEI, SBEIIa and SBEIIb classes. SBEIIa and SBEIIb generally exhibit around 80% sequence identity to each other, particularly in the central regions of the genes.
SBEIIa, SBEIIb and SBEI may also be distinguished by their expression patterns, both temporal and spatial, in endosperm and in other tissues. SBEI is expressed from mid-endosperm development onwards in wheat and maize (Morell et al., 1997). In contrast, SBEIIa and SBEIIb are expressed from an early stage of endosperm development. In maize, SBEIIb, is the predominant form in the endosperm whereas SBEIIa is present at high expression levels in the leaf (Gao et al., 1997). In rice, SBEIIa and SBEIIb are found in the endosperm in approximately equal amounts (Yamanouchi and Nakamura, 1992). However, there were differences in timing and tissues of expression. SBEIIa is expressed at an earlier stage of seed development, being detected at 3 days after flowering (DAF), and was expressed in leaves, while SBEIIb was not detectable at 3 DAF and was most abundant in developing seeds at 7-10 DAF and was not expressed in leaves (Mizuno et al., 2001). In wheat endosperm, SBEI (Morell et al, 1997) is found exclusively in the soluble fraction, while SBEIIa and SBEIIb are found in both soluble and starch-granule associated fractions (Rahman et al., 1995).
Two types of debranching enzymes are present in higher plants and are defined on the basis of their substrate specificities, isoamylase type debranching enzymes, and pullulanase type debranching enzymes (Myers et al., 2000). Sugary-1 mutations in maize and rice are associated with deficiency of both debranching enzymes (James et al., 1995, Kubo et al., 1999) however the causal mutation maps to the same location as the isoamylase-type debranching enzyme gene. In rice, antisense inhibition of isoamylase altered the structure of amylopectin and starch properties (Fujita et al., 2003), showing that isoamylase was required for amylopectin biosynthesis.
Representative starch branching enzyme genes that have been cloned from cereals are listed in Table 1.
TABLE 1Starch branching enzyme genes characterized from cereals including rice.SBEType ofSpeciesisoformcloneAccession No.ReferenceRiceSBEIcDNAD10752Nakamura andYamanouchi, 1992SBEIgenomicD10838Kawasaki et al., 1993RBE3cDNAD16201Mizuno et al., 1993RBE4cDNAAB023498Mizuno et al., 2001MaizeSBEIcDNAU17897Fisher et al., 1995genomicAF072724Kim et al., 1998aSBEIIbcDNAL08065Fisher et al., 1993genomicAF072725Kim et al., 1998SBEIIacDNAU65948Gao et al., 1997WheatSBEIIcDNAY11282Nair et al., 1997SBEIcDNA andAJ237897 SBEI gene)Baga et al., 1999genomicAF002821 (SBEI pseudogeneRahman et al., 1997,AF076680 (SBEI gene)Rahman et al., 1999AF076679 (SBEI cDNA)SBEIcDNAY12320Repellin et al., 1997SBEIIacDNA andAF338432 (cDNA)Rahman et al., 2001genomicAF338431 (gene)SBEIIbcDNA andWO 01/62934genomicSBEIIbcDNAWO 00/15810BarleySBEIIa andcDNA andAF064563 (SBEIIb gene)Sun et al., 1998SBEIIbgenomicAF064561 (SBEIIb cDNA)AF064562 (SBEIIa gene)AF064560 (SBEIIa cDNA)
In maize and rice, high amylose phenotypes have been shown to result from lesions in the SBEIIb gene, also known as the amylose extender (ae) gene (Boyer and Preiss, 1981, Mizuno et al., 1993; Nishi et al., 2001). In these SBEIIb mutants, endosperm starch grains showed an abnormal morphology, amylose content was significantly elevated, the branch frequency of the residual amylopectin was reduced and the proportion of short chains (<DP17, especially DP8-12) was lower. Moreover, the gelatinisation temperature of the starch was increased. In addition, there was a significant pool of material that was defined as “intermediate” between amylose and amylopectin (Boyer et al., 1980, Takeda, et al., 1993b). In rice, inactivation of SBEIIb led to an amylose content of about 25% compared to wild-type rice which has about 18% amylose (Nishi et al., 2001).
In contrast, maize plants mutant in the SBEIIa gene due a mutator (Mu) insertional element and consequently lacking in SBEIIa protein expression were indistinguishable from wild-type plants in the branching of endosperm starch (Blauth et al., 2001), although they were altered in leaf starch. Similarly, rice plants deficient in SBEIIa activity exhibited no significant change in the amylopectin chain profile in endosperm (Nakamura 2002). In both maize and rice, the SBEIIa and SBEIIb genes are not linked in the genome.
Very high amylose varieties of maize have been known for some time. LAPS (low amylopectin starch) maize which contains very high amylose content (>90%) was achieved by a considerable reduction in the SBEI activity together with an almost complete inactivation of SBEII activity (Sidebottom et al., 1998).
In potato, down regulation of the main SBE in tubers (SBE B, equivalent to SBEI) by antisense methods resulted in some novel starch characteristics but did not alter the amylose content (Safford et al., 1998). Antisense inhibition of the less abundant form of SBE (SBE A, analogous to SBEII in cereals) resulted in a moderate increase in amylose content to 38% (Jobling et al., 1999). However, the down regulation of both SBEII and SBEI gave much greater increases in the relative amylose content, to 60-89%, than the down-regulation of SBEII alone (Schwall et al., 2000).
In wheat, a mutant entirely lacking the SGP-1 (SSII) protein was altered in amylopectin structure and had deformed starch granules and an elevated amylose content to about 30-37% of the starch, which was an increase of about 8% over the wild-type level (Yamamori et al., 2000). Amylose was measured by colorimetric measurement, amperometric titration (both for iodine binding) and a concanavalin A method. Starch from the SSII null mutant exhibited a decreased gelatinisation temperature compared to starch from an equivalent, non-mutant plant. Starch content of the grain was reduced from 60% in the wild-type to below 50%.
In maize, the dull1 mutation causes decreased starch content and increased amylose levels in endosperm, with the extent of the change depended on the genetic background, and increased degree of branching in the remaining amylopectin (Shannon and Garwood, 1984). The gene corresponding to the mutation was identified and isolated by a transposon-tagging strategy using the transposon mutator (Mu) and shown to encode the enzyme designated starch synthase II (SSII) (Gao et al., 1998). The enzyme is now recognized as a member of the SSIII family in cereals (Li et al., 2003). Mutant endosperm had reduced levels of SBEIIa activity associated with the dull1 mutation. It is not known if these findings are relevant to other cereals, for example rice.
Lines of barley having an elevated proportion of amylose in grain starch have been identified. These include High Amylose Glacier (AC38) which has a relative amylose content of about 45%, and chemically induced mutations in the SSIIa gene of barley which raised levels of amylose in kernel starch to about 65-70% (WO 02/37955 A1; Morel et al., 2003). The starch showed reduced gelatinisation temperatures.
Rice (Oryza sativa L.) is the most important cereal crop in the developing world and is grown widely, particularly in Asia which produces about 90% of the world total.
Starch is widely used in the food, paper and chemical industries. The physical structure of starch can have an important impact on the nutritional and handling properties of starch for food or non-food or industrial products. Certain characteristics can be taken as an indication of starch structure including the distribution of amylopectin chain length, the degree and type of crystallinity, and properties such as gelatinisation temperature, viscosity and swelling volume. Changes in amylopectin chain length may be an indicator of altered crystallinity, gelatinisation or retrogradation of the amylopectin.
Starch composition, in particular the form called resistant starch which may be associated with high amylose content, has important implications for bowel health, in particular health of the large bowel. Accordingly, high amylose starches have been developed in certain grains such as maize and barley for use in foods as a means of promoting bowel health. The beneficial effects of resistant starch result from the provision of a nutrient to the large bowel wherein the intestinal microflora are given an energy source which is fermented to form inter alia short chain fatty acids. These short chain fatty acids provide nutrients for the colonocytes, enhance the uptake of certain nutrients across the large bowel and promote physiological activity of the colon. Generally if resistant starches or other dietary fiber is not provided the colon is metabolically relatively inactive.
Whilst chemically or otherwise modified starches can be utilised in foods that provide functionality not normally afforded by unmodified sources, such processing has a tendency to either alter other components of value or carry the perception of being undesirable due to processes involved in modification. Therefore it is preferable to provide sources of constituents that can be used in unmodified form in foods.
Therefore, rice having starch with a proportion of amylose greater than 40% is unknown. Although high amylose maize and barley varieties are known, very high amylose rice is preferred for rice growing regions. Starch from such rice is relatively resistant to digestion and therefore very high amylose rice is expected to bring an important health benefit to a substantial portion of the world population.
General
Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. The references mentioned herein are hereby incorporated by reference in their entirety. Reference herein to prior art, including any one or more prior art documents, is not to be taken as an acknowledgment, or suggestion, that said prior art is common general knowledge in Australia or forms a part of the common general knowledge in Australia.
As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source.
The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents Thymidine.